Cancer is a major disease burden and one hope of changing this is to develop new treatments through a[unreadable] better understanding of the disease. Recent discoveries concerning the activities of short RNAs in mammals[unreadable] may provide both new insights and new treatments of cancer. Cancer is a disease of gene dysregulation[unreadable] caused by mutations and epigenetic changes. Short RNAs are now known to regulate genes at the levels of[unreadable] mRNA degradation, primarily by siRNAs, mRNA translation, primarily by microRNAs, and mRNA[unreadable] transcription, primarily by repeat-associated short interfering RNAs (rasiRNAs). Recent results from[unreadable] bioinformatic studies indicate that approximately 20% of all mammalian mRNAs are probably regulated by[unreadable] mi RNAs. Furthermore, there is strong and rapidly growing evidence suggesting that changes in miRNA[unreadable] regulation is related to malignant transformation and in fact could be a critical event in oncogenic[unreadable] transformation. Little is known about the potential roles of short RNAs in silencing transcription at the level[unreadable] of chromatin in these cells. It is possible that some of the epigenetic changes and genomic instability[unreadable] common of cancers could be directed by RNAi-related pathways. Part of the revolution of RNA interference[unreadable] is the ability to express short hairpin RNAs from vectors to generate siRNAs which silence a specific gene.[unreadable] First, in collaboration with the Jacks and Lees projects, we will develop lentivirus vectors expressing shRNAs[unreadable] in a regulated fashion using tet-activated Pol II transcription. Second, we will use a very sensitive cloning[unreadable] technology for short RNAs to analyze their expression in T-cell populations as they undergo development.[unreadable] The activities of specific miRNAs will be related to known developmental transitions in this defined pathway.[unreadable] Furthermore, the nature of short RNAs in T-cell lymphomas and other tumor cells will be investigated by[unreadable] collaboration with the other projects. The silencing of repetitive sequences by both chromatin modification[unreadable] and DNA methylation is frequently observed in embryonic stem (ES) cells. We will clone short RNAs from[unreadable] ES cells which have been induced to express high levels of RNA from repetitive sequences to investigate[unreadable] the role of short RNAs in gene silencing at the level of transcription. The relationship between the[unreadable] resilencing of repetitive and unique sequences in these ES cells and the sequences of cloned short RNAs[unreadable] will be investigated. Furthermore, the dependence of these processes on Dicer, Argonaute and other RNAi-related[unreadable] genes will be determined. We will also determine the function of a cluster of miRNAs that is[unreadable] exclusively expressed in ES cells and embryonic tissue. Finally, we have found that both the retinoblastoma[unreadable] (Rb) pathway and the RNAi pathway regulate post-mitotic nuclear division in the intestinal epithelium of C.[unreadable] elegans. We will investigate whether related pathways are important for regulation of cell division in[unreadable] mammalian cells.[unreadable]